Recent observations concerning the limits of tolerance of the brain to anoxia and ischemia and current speculation over the role of oxygen free radicals in the evolution of irreversible neuronal injury have focused increased attention upon the regulation of mitochondrial oxidative phosphorylation in general, and the effects of these insults upon cerebral mitochondrial behavior in particular. Among available techniques, only cortical microfluorometry permits continuous, non-destructive analysis of the cerebral mitochondrial redox state during metabolic perturbations. However, traditional microfluorometric method for measuring cortical mitochondrial respiration in vivo is seriously compromised by hemodynamic and oximetric artifacts. We have recently developed a technique, isosbestic fluorometry, which provides appropriate correction for these vascular phenomena and permits simultaneous evaluation of mitochondrial NADH:NAD plus redox state, microcirculatory volume, and hemoglobin oxygenation. Furthermore, we believe that isosbestic fluorometry provides, for the first time, quantitative metabolic information. We propose to employ isosbestic fluorometry in studies of canine cerebral cortex designed to confirm (or refute) the quantitative nature of the technique; to examine in detail a remarkable phenomenon observed in preliminary studies of cerebral anoxia, i.e., transient oxidation of NADH; to employ selected metabolic inhibitors and uncouplers to elucidate mechanisms whereby mitochondrial respiration is regulated during anoxia, graded hypoxia, and ischemia; and to evaluate, where appropriate, metabolic interventions (e.g., barbiturate therapy) purported to protect the brain during anoxia and ischemia with respect to their effects upon mitochondrial respiration.